Rediscovering the Actin Cytoskeleton

New techniques leading to discoveries in cell migration

The actin cytoskeleton of the cell forms a rigid network of interconnected cables that provide force and mechanical support to maintain cell shape (Figure 1). However, the actin cytoskeleton is not simply a static compilation of rigid filaments that dictate cell morphology. The cytoskeleton has an inherent plasticity that stems from the ability of actin to polymerize and depolymerize in response to extracellular cues. In fact, it is both a dynamic support and signaling scaffold that receives and transmits complex mechanosensing and signal transduction stimuli important for many fundamental cellular functions, including axon pathfinding, cell migration, differentiation, and proliferation. On the other hand, when deregulated, aberrations in the cytoskeleton contribute to numerous human diseases, including developmental disorders, chronic inflammation, and cancer.

The processes of cell migration and cancer metastasis provide an interesting example of how the actin cytoskeleton controls normal and diseased cell translocation. The actin cytoskeleton and associated regulatory proteins are distinctly polarized in migrating cells to form a leading front and trailing rear compartment (Figure 2). At the front of the cell, actin polymerization and assembly drive membrane protrusion, leading to the formation of filopodia and the lamellipodium. At the sides and rear of the cell, cortical actin provides cell rigidity, while stress fibers work in concert with myosin to facilitate strong force and contraction events that pull up the trailing tail from the underlying substratum. The repeated cycle of membrane extension at the front and tail retraction at the back facilitates cell translocation.

LEADING EDGE

Actin Polymerization

and Depolymerization

Focal Adhesion

Stress Fibers

Lamellipodium

Direction of

Direction of

Migration

Migration

TAIL

Figure 2. Schematic of a polarized migrating cell showing the organization of the actin cytoskeleton and focal adhesions around the leading edge and the trailing tail.

The fundamental ability of cells to migrate is important for normal tissue patterning during embryonic development, wound healing, and immune cell trafficking functions. However, most cells in the adult organism are stationary, as they are statically associated with specialized tissues and organ structures. For reasons that are not yet clear, proliferating cancer cells can aberrantly turn on migration signals that activate the actin cytoskeleton machinery to drive cell locomotion. These motile cells are highly dangerous, because they can now leave the primary tumor site, invade through the surrounding tissues, and gain access to the circulation, where they travel to distant organs and form secondary metastatic tumors. Unfortunately, there are no therapeutic treatments available that target migrating cancer cells, and thus, the majority of patients with metastatic cancer succumb to the disease.

The example of migrating cancer cells illustrates the need to understand precisely how the actin cytoskeleton is regulated in normal and diseased cells. Traditional methods for studying the actin cytoskeleton and its associated regulatory partners involve examining individual, chemically fixed cells using immunofluorescence and green fluorescent protein

(GFP)-based technologies, along with fluorescent microscopy. This basic approach has revealed a number of key regulatory components and their spatial organization within the interior of the cell. However, visualization of structures alone does not address how the actin scaffold and its numerous components are functionally integrated to drive specific cellular changes. Understanding the actin regulatory network as a system can help identify functional protein units that operate together in a defined space within the cell and help identify where and how certain multi- component scaffolding interactions occur.

interactions, or cellular consequences of silencing specific genes. To achieve these goals, it is necessary to fully define the actin cytoskeleton proteome and phosphoproteome in detail. New techniques, coupled with powerful informatics programs and computer models can then be used to understand how complex protein-protein interactions, signal transduction events, and kinase/phosphatase activation networks are differentially integrated in normal and diseased cells.

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Perspectives on new techniques

There is a central need to better define the actin cytoskeleton proteome, understand how the nuts and bolts of the actin domain are assembled into a functional cytoskeletal network, and show how the domain is integrated with the other cellular domains that comprise a complete functioning cell. However, studying the proteins that regulate the cytoskeleton has proven difficult because the F-actin cytoskeleton is insoluble in detergents like Triton®-X100. Also, many proteins/phosphoproteins, upon activation, move from the soluble cytoplasmic compartment to the insoluble

cytoskeleton. This has made it difficult to study their biochemical changes, such as phosphorylation and nitrosylation. The need to study the molecular composition and biochemical modifications of the actin domain has spurred the development of a new cytoskeleton purification method that enables the enrichment of cytoskeleton-associated proteins in their native state (Figure 3). Western blot analysis (Figure 4) illustrates the robust purification and enrichment of the known cytoskeleton proteins, actin, a-actinin, Src, and vinculin, using this technique.

Microscopic analysis of the purified cytoskeleton shows that the actin-associated protein, a-actinin remains bound to the native, unperturbed, F-actin cytoskeleton after extraction of the soluble cytoplasmic proteins (Figure 5). The extraction of cytoplasmic proteins also enhances the ability to specifically visualize actin-associated proteins like a-actinin by reducing nonspecific background noise (Figure 5, right column). This increase in signal-to-noise substantially enhances the detection and localization of low-abundance proteins that associate with the actin cytoskeleton (Figure 6).

Figure 5. a-actinin remains strongly bound to the actin cytoskeleton after the cytoskeleton enrichment procedure and is more clearly visualized after removal of soluble

background proteins. Fluorescent photomicrographs of mouse embryonic fibroblasts co-expressing mCherry-tagged actin (red) and the actin binding and regulatory protein a-actinin, which is tagged with GFP (green). Images were captured from a live cell before and after extraction of soluble cytoplasmic proteins with the EMD Millipore ProteoExtract® Native Cytoskeleton Enrichment and Staining Kit (Catalogue No.

Finally, quantitative protein profiling of enriched cytoskeletons by mass spectrometry and functional annotation of these 998 identified proteins reveals that many known cytoskeleton, focal adhesion, and actin signaling proteins are present in the cytoskeleton fraction. These proteins include myosin isoforms and filamin-B (Figure 7 & Table I, which shows a representative list of the 998 identified International Protein Index (IPI) proteins). Also, this new cytoskeleton enrichment procedure enabled the identification of a large number of unique peptides and provided excellent amino acid sequence coverage for each protein.

Figure 7. Mass spectrometry analysis of cytoskeleton- enriched proteins. Chart showing the percentage of cytoskeleton-enriched proteins annotated according to their known functions using the Ingenuity Knowledge Database. 698 proteins were annotated from a total of 800 proteins identified using the EMD Millipore ProteoExtract® Cytoskeleton Enrichment and Isolation Kit (Catalogue No. 17-10195) and mass spectrometry.

Table 1. Top matched proteins from the isolated CSK based on the number of spectral count. Representative list of the 998 identified International Protein Index (IPI) proteins).

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Novel techniques for purifying the cytoskeleton will be useful tools for studying the protein composition of the actin cytoskeleton under a wide range of cellular conditions and disease states, including migration and cancer cell metastasis. For example, the new cytoskeleton enrichment procedure, combined with a new technique to simultaneously activate cell migration, enabled the comparison of cytoskeleton proteome compositions of static versus migrating cells. A newly developed Cell Comb™ (EMD Millipore, Catalogue No. 17-10191, Figure 8) was used to efficiently and rapidly introduce a large number of precisely defined wounds through a confluent cell monolayer (Figure 9). As a result, significant numbers of cells were induced to migrate synchronously to fill in the wounded area (Figure 10).

Figure 8. EMD Millipore’s

Cell Comb™ (Catalogue No.

17-10191).

One Direction

Two Directions

Figure 9. Uniform scratches created by the scratch assay Cell Comb™ device. Scratches produced by the EMD Millipore Cell Comb™ for Scratch Assay (Catalogue No. 17-10191) in one direction (parallel lines) or two directions (checkered pattern) allow for higher density in wound generation within the provided rectangular culture plate.

The ability to simultaneously activate large numbers of migrating cells facilitated temporal analysis of the signal transduction process that governs cell migration (Figure 11). It is also important for biochemical and proteomic methods that require substantial amounts of protein for quantitative analyses. Assaying large numbers of cells is especially important when profiling subcellular fractions, like the cytoskeleton, which typically results in reduced yields of protein.

Therefore, the robust wounding approach described was combined with the ability to enrich the actin cytoskeleton in its native configuration from migrating or stationary cells. Cells were induced to migrate for 20 h before their cytoskeletons were enriched and examined using mass spectrometry. From a total of 800 cytoskeleton-associated proteins identified, more than 80 unique proteins showed a 1.5 fold increase in the cytoskeleton fraction purified from migrating cells compared with cytoskeletons isolated from non-motile cells (Table 2 shows a representative list of these 80 proteins). In contrast, 25 cytoskeleton proteins showed a 1.5 decrease in migrating cells when compared to cytoskeletons purified from stationary cells (Table 2). These findings illustrate the power of these technologies to identify proteins that regulate the cytoskeleton in migrating cells. A similar approach could be used to profile the cytoskeletons from migrating, invasive cancer cells to identify biomarkers that are prognostic and diagnostic of metastatic disease.

A.

Wound Minutes

None

0

30

60

120

240

p-FAK

p130CAS

p-ErK

ErK

GAPDH

B.

Wound Minutes

None

0

30

60

120

240

GTP-Rac1

Figure 11. Activation of

signaling molecules involved

in cell migration. NIH 3T3 cell monolayers were scratched using the Cell Comb™ for Scratch Assay (Catalogue No. 17-10191) and whole cell lysates were prepared at 0, 30, 60, 120, 240 minutes, post-wounding. (A) Western blot detection of p-FAK, CAS, p-Erk and Erk ; GAPDH is used as a loading control. (B) Time dependent activation of Rac1 using a Rac1 activity assay kit (IP followed by WB). High density and uniformity of the scratches produced by the Cell Comb™ for Scratch Assay allows for the detection of changes in protein activation via Western blot,

The development of robust and easy-to-handle cytoskeleton enrichment assays, combined with cell-based tools for biological analyses, opens the door to identifying and studying the components of the actin cytoskeleton in unprecedented detail. This and the fact that these emerging technologies are amenable to large-scale biochemical and proteomic studies will facilitate a comprehensive understanding of how the cytoskeleton domain functions under a wide range of physiological and disease conditions. Future work in this area can provide a functional blueprint of the cytoskeleton machinery. Once this is achieved, it may be possible to design specific therapeutics that target the cytoskeleton in diseased cells. The possible health benefits are far-reaching and the future for cytoskeleton research has never looked brighter.